Abstract: Nearshore microbial communities serve as critical bioindicators of marine ecosystem health. However, their biogeographical patterns and the specific distribution of pathogenic Vibrio have not been fully characterized. In this study, surface seawater samples collected from the nearshore waters of Hainan Island were analyzed using high-throughput sequencing and multivariate statistical methods to investigate the spatial differentiation of microbial communities and their relationships with salinity and pH. A total of 37 phyla and 81 classes were identified across 31 sampling sites. Principal Coordinate Analysis (PCoA) combined with K-means clustering showed that microbial communities in the nearshore waters of Hainan Island exhibited significant spatial differentiation and could be classified into two groups. Group I was mainly distributed in the southern nearshore waters and was characterized by the enrichment of Vibrionaceae and Pseudoalteromonadaceae. Group II was mainly distributed in the northern nearshore waters, showed relatively higher community diversity than Group I, and was significantly enriched in Cyanobiaceae and Rhodobacteraceae. Environmental association analysis indicated that while salinity gradients statistically explained the spatial differentiation, the distribution characteristics of autotrophic and heterotrophic functional groups suggest that nutrient levels, such as organic load, may be one of the important core drivers reshaping community structure. In addition, different potentially pathogenic Vibrio lineages exhibited distinct habitat preferences. The southern nearshore fishing ports and aquaculture areas were characterized by the enrichment of Harveyi clade, indicating that such habitats have certain relevance to aquaculture health concerns. This study reveals the spatial distribution patterns of nearshore microbial communities and potentially pathogenic Vibrio under the influence of environmental gradients in a tropical island setting, and provides a scientific reference for studies on nearshore environmental management and aquaculture disease prevention.